Polymer factor VIII moiety conjugates

ABSTRACT

Conjugates of a Factor VIII moiety and one or more water-soluble polymers are provided. Typically, the water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided are compositions comprising the conjugates, methods of making the conjugates, and methods of administering compositions comprising the conjugates to a patient.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/702,302, now U.S. Pat. No. 7,858,749, filed Feb. 5, 2007, which is acontinuation of U.S. patent application Ser. No. 10/789,956, now U.S.Pat. No. 7,199,223, filed Feb. 26, 2004, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 60/450,578, filedFeb. 26, 2003, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to conjugates comprising aFactor VIII moiety (i.e., a moiety having Factor VIII activity) and apolymer. In addition, the invention relates to compositions comprisingthe conjugates, methods for synthesizing the conjugates, and methods fortreating patients.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Dec. 11, 2009 and named“417148301US05seqlist.txt” (40918 bytes), the contents of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Hemostasis is the process of arresting the outflow of blood from aninjured blood vessel. For mammals, as well as many other organisms, thehemostatic process is critically important for continued survival.Defects in the hemostatic process can result in, for example, theinability to effectively form blood clots that serve to stop the loss ofblood following vascular injury. In humans, individuals who suffer froman inability to form blood clots are called hemophiliacs. Of particularconcern for hemophiliacs is the life-threatening risk that once started,bleeding will never cease.

Generally, hemophiliacs lack the ability to produce effective amounts ofone or more substances required to form blood clots. For example,hemophiliacs who suffer from hemophilia A (also called “classichemophilia”) have an inability to produce effective levels of FactorVIII (also known as “antihemophilia factor A,” “antihemophilicglobulin,” and “AHG”). Factor VIII is a key component of one of several“cascades” of reactions that result in the formation of blood clots.Critical for the cascade of reactions referred to as the “intrinsicpathway,” Factor VIII ultimately influences the conversion of fibrinogeninto the major component of blood clots, fibrin.

Although the intrinsic pathway of blood clot formation is relativelycomplex, the role of Factor VIII can be described briefly. In thepresence of relatively small amounts of thrombin (released, for example,by the cells of ruptured tissues), Factor VIII is converted into itsactivated form known as Factor VIIIa. Factor VIIIa (along with othersubstances), in turn, activates another factor, Factor X into Factor Xa.Thereafter, Factor Xa (along with other substances) converts prothrombininto thrombin, with the result that a relatively large amount ofthrombin is produced over time. Relatively large amounts of thrombineffectively convert fibrinogen into fibrin. Fibrin, in turn, forms thematrix or lattice responsible for the formation of blood clots. FactorVIII's role in the intrinsic pathway of blood clotting is shownschematically below.

Affecting one or two males for every 10,000 live births in allpopulations, hemophilia A can result from any one of a variety ofmutations of the Factor VIII gene, which is located on the X-chromosome.Depending on the particular mutation, hemophilia A can manifest itselfas severe, moderate or mild. Individuals suffering from the severestforms of hemophilia A entirely lack the ability to express active formsof Factor VIII. Clinically, individuals affected with hemophilia Asuffer from muscle hemorrhage, joint hemorrhage, easy bruising, andprolonged bleeding from wounds. The mean age of individuals sufferingfrom hemophilia A without treatment is twenty. Current treatment ofhemophilia A involves the infusion of exogenous Factor VIII concentratecollected from human plasma or prepared via recombinant DNA techniques.Because these treatments serve only to supplement the lack of effectivelevels of Factor VIII, individuals suffering from Factor VIII requireregular injections of Factor VIII concentrate throughout their lives.

Several commercial forms of Factor VIII concentrates are available toprovide replacement therapy for patients suffering from hemophilia A.For example, blood-derived Factor VIII concentrate products are soldunder the HEMOFIL® M, Antihemophilic Factor VIII (human) (Baxter,Deerfield, Ill.), KOATE® DVI, Antihemophilic Factor VIII (human) (Bayer,Research Triangle Park, N.C.), MONARC-M™, Antihemophilic Factor VIII(human) (American Red Cross, Washington, D.C.), and MONOCLATE P®,Antihemophilic Factor VIII (human) (Aventis, Bridgewater, N.J.) brands.With respect to recombinantly prepared Factor VIII concentrates,commercial products are provided under the HELIXATE® FS, Factor VIII,recombinant (Aventis, Bridgewater, N.J.), KOGENATE FS®, Factor VIII,recombinant (Bayer, Research Triangle Park, N.C.), RECOMBINATE®, FactorVIII, recombinant (Baxter, Deerfield, Ill.), ADVATE®, Factor VIII,recombinant (Baxter, Deerfield, Ill.), and REFACTO®, Factor VIII,recombinant (Wyeth/Genetics Institute, Cambridge, Mass.) brands.

Generally, recombinant sources of Factor VIII concentrates are favoredover blood-derived sources since the latter involves the risk oftransmitting viruses and/or other diseases associated with blooddonation. While recombinant-based formulations avoid these drawbacks,the processing of recombinant-based products often requires the presenceof certain proteins such as albumin, which are inevitably present in thefinal formulation administered to the patient. Often, patients whoreceive such formulations develop allergic reactions to these foreignproteins. In any event, both blood-derived and recombinant-basedproducts suffer from the disadvantage of repeated administration.

PEGylation, or the attachment of a poly(ethylene glycol) derivative to aprotein, has been described as a means to reduce immunogenicity as wellas a means to prolong a protein's in vivo half-life. With respect toFactor VIII, however, previous experiences with forming protein-polymerconjugates has proven to be of little predictive value vis-à-vis polymercoupling to Factor VIII. See U.S. Pat. No. 4,970,300.

Notwithstanding these difficulties, attempts of preparing satisfactorycompositions of conjugates of certain polymers to Factor VIII have beendescribed. For example, previously referenced U.S. Pat. No. 4,970,300describes the PEGylation of Factor VIII using a specific poly(ethyleneglycol) derivative having a molecular weight within the range of about500 to 5,000. In addition, U.S. Pat. No. 6,048,720 describes efficientprotection against degradation in an in vitro environment when four tofive monomethoxy poly(ethylene glycol) strands are conjugated to FactorVIII.

None of these described conjugates, however, has proven tosatisfactorily address the problems associated with current FactorVIII-based therapies. For example, conjugates comprised of relativelysmall polymers (e.g., of about 5,000 Daltons or less) may not suitablyprovide extended in vivo half-life and/or sufficiently reduced immuneresponse. In addition, conjugates having many individual polymersattached to Factor VIII are more likely to have reduced activity as aresult of the polymer(s) blocking sites necessary for activity.

Thus, there remains a need in the art to provide additional conjugatesbetween water-soluble polymers and moieties having Factor VIII activity.The present invention is therefore directed to such conjugates as wellas compositions comprising the conjugates and related methods asdescribed herein, which are believed to be new and completelyunsuggested by the art.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of this invention to provide acomposition comprising a plurality of conjugates, preferably althoughnot necessarily, each having one to three water-soluble polymerscovalently attached to a Factor VIII moiety, wherein each water-solublepolymer preferably has a nominal average molecular weight in the rangeof greater than 5,000 Daltons to about 100,000 Daltons.

It is another object of the invention to provide such a conjugatewherein each of the water-soluble polymers is a poly(alkylene oxide).

It is an additional object of the invention to provide such a conjugatewherein each of the water-soluble polymers is a poly(ethylene glycol).

It is a further object of the invention to provide a conjugatecomprising a plurality of monoPEGylated Factor VIII moiety conjugates.

It is still a further object of the invention to provide a method forpreparing polymer conjugates comprising the steps of contacting one ormore activated, water-soluble polymers to a Factor VIII moiety underconditions sufficient to result in a plurality of conjugates, preferablyalthough not necessarily, each having one to three water-solublepolymers covalently attached to a Factor VIII moiety, wherein thewater-soluble polymer preferably has a nominal average molecular weightin the range of greater than 5,000 Daltons to about 150,000 Daltons.

It is an additional object of the invention to provide a method fortreating a patient in need of Factor VIII therapy, comprising the stepof administering to the patient a composition as described herein,wherein the composition contains a therapeutically effective amount ofone or more of the conjugates.

It is still yet an additional object of the invention to provide amethod for preparing a water-soluble polymer-Factor VIII moietyconjugate comprising the step of contacting, under conjugationconditions, a Factor VIII moiety with a polymeric reagent.

Additional objects, advantages and novel features of the invention willbe set forth in the description that follows, and in part, will becomeapparent to those skilled in the art upon reading the following, or maybe learned by practice of the invention.

In one embodiment then, a composition is provided comprising a pluralityof conjugates, preferably although not necessarily, each having one tothree-water soluble polymers covalently attached to a Factor VIIImoiety, wherein each water-soluble polymer preferably has a nominalaverage molecular weight in the range of greater than 5,000 Daltons toabout 150,000 Daltons. Although any Factor VIII moiety can be used, itis preferred that the compositions comprise Factor VIII per se (asdescribed in, for example, U.S. Pat. No. 4,757,006), Factor VIIIa (i.e.,the activated form of Factor VIII produced when Factor VIII is placed incontact with relatively small amounts of thrombin), Factor VIII:vWF(i.e., Factor VIII bound to von Willebrand Factor), and/or truncatedversions of Factor VIII such as B-domain deleted Factor VIII (asdescribed in, for example, U.S. Pat. No. 4,868,112).

The polymer(s) can be any water-soluble polymer and the invention is notlimited in this regard. It is preferred, however, that each polymerpresent in the conjugate is selected from the group consisting ofpoly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol),polyoxazoline, poly(acryloylmorpholine), and combinations thereof. It isparticularly, preferred, however, that a poly(alkylene oxide) such as apoly(ethylene glycol) derivative is used as the polymer in the presentinvention.

The conjugates described herein advantageously reduce immunogenicity, aproblem encountered by many hemophiliacs treated with exogenous sourcesof Factor VIII. In addition, the present conjugates require decreasedfrequency of dosing compared to Factor VIII compositions lackingconjugates. By reducing the frequency of dosing, the conjugatesadvantageously decrease the number of painful injections hemophiliacsmust endure in order to provide sustained levels of an agent havingFactor VIII activity.

In another embodiment, a method for preparing a conjugate is provided.The method comprises the step of contacting one or more activated,water-soluble polymers (i.e., a polymeric reagent) preferably having anominal average molecular weight in the range of greater than 5,000Daltons to about 150,000 Daltons to a Factor VIII moiety. Activation ofthe water-soluble polymer can be accomplished under any art-known methodso long as the resulting polymer, under the proper conditions of pH,temperature, and so forth, will form a covalent bond such that theFactor VIII moiety is covalently attached to the polymer. Contacting ofthe one or more activated, water-soluble polymers to the Factor VIIImoiety is carried out under those conditions required for the activated,water-soluble polymer to form a covalent attachment at the desired sitein the moiety. The method results in a plurality of conjugates,preferably although not necessarily, each having one to threewater-soluble polymers covalently attached to the Factor VIII moiety. Insome instances, the conjugate can comprise a single polymer attached totwo, three, four, five, six, seven, eight, or more Factor VIII moieties.Optionally, the resulting composition can be further processed in orderremove undesired species such as, for example, conjugates having anundesired number of polymers. In order to remove such undesired species,purification techniques such as size-exclusion chromatography can beused.

In still another embodiment of the invention, compositions are providedcomprising a conjugate of the invention in combination with apharmaceutically acceptable excipient. The compositions encompass alltypes of formulations and in particular those that are suited forinjection such as powders that can be reconstituted, as well as liquids(e.g., suspensions and solutions).

In an additional embodiment of the invention, a method of administeringthe conjugate is provided. The method of administering comprises thestep of administering to the patient a composition as described herein,wherein the composition contains a therapeutically effective amount ofthe conjugate. Typically, the step of administering theconjugate-containing composition is effected by injection (e.g.,intramuscular injection, intravenous injection, subcutaneous injection,and so forth).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a SEC plot corresponding to the reaction mixture formed uponpegylation of B-Domain deleted Factor VIII with mPEG-SPA, 30K, asdescribed in Example 6.

FIG. 2 is a SEC plot corresponding to purified B-Domain Deleted FactorVIII-PEG conjugate prepared by conjugating the protein to mPEG-SPA, 30K,as described in Example 6.

FIG. 3 is SEC plot corresponding to the reaction mixture formed uponPEGylation of B-Domain deleted Factor VIII with mPEG-MAL, 20K, asdescribed in Example 7. As can be seen, the yield of monoPEGylatedproduct was approximately 33%.

FIG. 4 is a SEC plot corresponding to purified B-Domain Deleted FactorVIII-PEG conjugate prepared by conjugating the protein to mPEG-MAL, 20K,as described in Example 7. The purified conjugate was ˜94% PEG monomer.

FIG. 5 is a SEC plot corresponding to the reaction mixture formed uponPEGylation of B-Domain Deleted Factor VIII with mPEG-SMB, 30K, asdescribed in Example 8. The yield of monoPEGylated protein (1-mer) wasapproximately 41%.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to the particularpolymers, synthetic techniques, Factor VIII moieties, and the like, assuch may vary.

It must be noted that, as used in this specification and the intendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a polymer” includes a single polymer as well as two ormore of the same or different polymers, reference to a “an optionalexcipient” refers to a single optional excipient as well as two or moreof the same or different optional excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are interchangeable and meant to encompass any water-solublepoly(ethylene oxide). Typically, PEGS for use in accordance with theinvention comprise the following structure “—(OCH₂CH₂)_(n)—” where (n)is 2 to 4000. As used herein, PEG also includes“—CH₂CH₂—O(CH₂CH₂CH₂O)_(n)—CH₂CH₂—” and “—(OCH₂CH₂)_(n)O—,” dependingupon whether or not the terminal oxygens have been displaced. Throughoutthe specification and claims, it should be remembered that the term“PEG” includes structures having various terminal or “end capping”groups and so forth. The term “PEG” also means a polymer that contains amajority, that is to say, greater than 50%, of —OCH₂CH₂— repeatingsubunits. With respect to specific forms, the PEG can take any number ofa variety of molecular weights, as well as structures or geometries suchas “branched,” “linear,” “forked,” “multifunctional,” and the like, tobe described in greater detail below.

The terms “end-capped” and “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group, more preferably aC₁₋₁₀ alkoxy group, and still more preferably a C₁₋₅ alkoxy group. Thus,examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxyand benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. It must be remembered that the end-capping moiety may include oneor more atoms of the terminal monomer in the polymer [e.g., theend-capping moiety “methoxy” in CH₃(OCH₂CH₂)_(n)—]. In addition,saturated, unsaturated, substituted and unsubstituted forms of each ofthe foregoing are envisioned. Moreover, the end-capping group can alsobe a silane. The end-capping group can also advantageously comprise adetectable label. When the polymer has an end-capping group comprising adetectable label, the amount or location of the polymer and/or themoiety (e.g., active agent) to which the polymer is coupled can bedetermined by using a suitable detector. Such labels include, withoutlimitation, fluorescers, chemiluminescers, moieties used in enzymelabeling, colorimetric (e.g., dyes), metal ions, radioactive moieties,and the like. Suitable detectors include photometers, films,spectrometers, and the like. The end-capping group can alsoadvantageously comprise a phospholipid. When the polymer has anend-capping group comprising a phospholipid, unique properties areimparted to the polymer and the resulting conjugate. Exemplaryphospholipids include, without limitation, those selected from the classof phospholipids called phosphatidylcholines. Specific phospholipidsinclude, without limitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin.

“Non-naturally occurring” with respect to a polymer as described herein,means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer of the invention may, however, containone or more monomers or segments of monomers that are naturallyoccurring, so long as the overall polymer structure is not found innature.

The term “water soluble” as in a “water-soluble polymer” polymer is anypolymer that is soluble in water at room temperature. Typically, awater-soluble polymer will transmit at least about 75%, more preferablyat least about 95%, of light transmitted by the same solution afterfiltering. On a weight basis, a water-soluble polymer will preferably beat least about 35% (by weight) soluble in water, more preferably atleast about 50% (by weight) soluble in water, still more preferablyabout 70% (by weight) soluble in water, and still more preferably about85% (by weight) soluble in water. It is most preferred, however, thatthe water-soluble polymer is about 95% (by weight) soluble in water orcompletely soluble in water.

“Nominal average molecular weight” in the context of a water-soluble,non-naturally occurring polymer such as PEG, refers to the mass averagemolecular weight of the polymer, typically determined by size-exclusionchromatography, MALDI (matrix assisted laser desorption/ionization),light scattering techniques, or intrinsic velocity determination in1,2,4-trichlorobenzene. The polymers are typically polydisperse,possessing low polydispersity values of preferably less than about 1.2,more preferably less than about 1.15, still more preferably less thanabout 1.10, yet still more preferably less than about 1.05, and mostpreferably less than about 1.03.

The term “active” or “activated” when used in conjunction with aparticular functional group, refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to reacti.e., a “non-reactive” or “inert” group).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof as well as unprotected forms.

The terms “linkage” or “linker” are used herein to refer to an atom or acollection of atoms optionally used to link interconnecting moietiessuch as a terminus of a polymer segment and a Factor VIII moiety or anelectrophile or nucleophile of a Factor VIII moiety. The linker of theinvention may be hydrolytically stable or may include a physiologicallyhydrolyzable or enzymatically degradable linkage.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to15 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl as well as cycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or Spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8carbon atoms. “Cycloalkylene” refers to a cycloalkyl group that isinserted into an alkyl chain by bonding of the chain at any two carbonsin the cyclic ring system.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, and soforth).

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenoninterfering substituents, such as, but not limited to: alkyl, C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. “Substituted aryl” is aryl having oneor more noninterfering groups as a substituent. For substitutions on aphenyl ring, the substituents may be in any orientation (i.e., ortho,meta, or para).

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, or nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom that is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from noninterfering substituents.

“Electrophile” and “electrophilic group” refer to an ion or atom orcollection of atoms, that may be ionic, having an electrophilic center,i.e., a center that is electron seeking, capable of reacting with anucleophile.

“Nucleophile” and “nucleophilic group” refers to an ion or atom orcollection of atoms that may be ionic having a nucleophilic center,i.e., a center that is seeking an electrophilic center or with anelectrophile.

A “physiologically cleavable” or “hydrolysable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. Preferred are bonds that have a hydrolysis half-life at pH8, 25° C. of less than about 30 minutes. The tendency of a bond tohydrolyze in water will depend not only on the general type of linkageconnecting two central atoms but also on the substituents attached tothese central atoms. Appropriate hydrolytically unstable or weaklinkages include but are not limited to carboxylate ester, phosphateester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines,orthoesters, peptides and oligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, but are not limited to, thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Pharmaceutically acceptable excipient or carrier” refers to anexcipient that may optionally be included in the compositions of theinvention and that causes no significant adverse toxicological effectsto the patient. “Pharmacologically effective amount,” “physiologicallyeffective amount,” and “therapeutically effective amount” are usedinterchangeably herein to mean the amount of a polymer-Factor VIIImoiety conjugate that is needed to provide a desired level of theconjugate (or corresponding unconjugated Factor VIII moiety) in thebloodstream or in the target tissue. The precise amount will depend uponnumerous factors, e.g., the particular Factor VIII moiety, thecomponents and physical characteristics of the therapeutic composition,intended patient population, individual patient considerations, and thelike, and can readily be determined by one skilled in the art, basedupon the information provided herein.

“Multi-functional” means a polymer having 3 or more functional groupscontained therein, where the functional groups may be the same ordifferent. Multi-functional polymeric reagents of the invention willtypically contain from about 3-100 functional groups, or from 3-50functional groups, or from 3-25 functional groups, or from 3-15functional groups, or from 3 to 10 functional groups, or will contain 3,4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone.

The term “Factor VIII moiety,” as used herein, refers to a moiety havingFactor VIII activity. The Factor VIII moiety will also have at least oneelectrophilic group or nucleophilic group suitable for reaction with apolymeric reagent. Typically, although not necessarily, the Factor VIIImoiety is a protein. In addition, the term “Factor VIII moiety”encompasses both the Factor VIII moiety prior to conjugation as well asthe Factor VIII moiety residue following conjugation. As will beexplained in further detail below, one of ordinary skill in the art candetermine whether any given moiety has Factor VIII activity.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of anactive agent (e.g., conjugate), and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutarnine is Gln or Q; Asparagine is Asnor N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid isGlu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Argor R; and Glycine is Gly or G.

Turning to a first embodiment of the invention then, a composition isprovided comprising a plurality of conjugates, preferably although notnecessarily, each having one to three water-soluble polymers covalentlyattached to a Factor VIII moiety, wherein each of the water-solublepolymers preferably has a nominal average molecular weight in the rangeof greater than 5,000 Daltons to about 150,000 Daltons.

Native Factor VIII is a 2,351 amino acid, single chain glycoprotein thatis structurally organized as A1-A2-B-A3-C1-C2. The expressed 2,351 aminoacid sequence is provided as SEQ ID NO:1. When the expressed polypeptideis translocated into the lumen of the endoplasmic reticulum, however, a19-amino acid signal sequence is cleaved, resulting in a secondsequence. This second sequence, herein provided as SEQ ID NO:2, lacksthe leading 19 amino acids is conventionally used by researchers toassign a numeric location (e.g., Arg³⁷²) to a given amino acid residueof Factor VIII. Thus, unless specifically noted, all assignments of anumeric location of an amino acid residue as provided herein are basedon SEQ ID NO:2.

In the presence of relatively small amounts of thrombin, Factor VIII iscleaved by thrombin at Arg³⁷², Arg⁷⁴⁰, and Arg¹⁶⁸⁹ to produce FactorVIIIa. Factor VIIIa is a heterotrimer comprised of the A1 subunit bound(via a copper ion) to the thrombin-cleaved light chain A3-C1-C2 and thefree A2 subunit bound via ionic interactions to A1. It will beappreciated that a Factor VIII moiety is not limited to merely “active”forms of Factor VIII (e.g., Factor VIIIa) and that the term “Factor VIIImoiety” encompasses “precursor” forms as well as other substances thathaving a similar procoagulant effect.

For any given moiety, it is possible to determine whether that moietyhas Factor VIII activity. For example, several animal lines have beenintentionally bred with the genetic mutation for hemophilia such that ananimal produced from such a line has very low and insufficient levels ofFactor VIII. Such lines are available from a variety of sources such as,without limitation, the Division of Laboratories and Research, New YorkDepartment of Public Health, Albany, N.Y. and the Department ofPathology, University of North Carolina, Chapel Hill, N.C. Both of thesesources, for example, provide canines suffering from canine hemophiliaA. In order to test the Factor VIII activity of any given moiety inquestion, the moiety is injected into the diseased animal, a small cutmade and bleeding time compared to a healthy control. Another methoduseful for determining Factor VIII activity is to determine cofactor andprocoagulant activity. See, for example, Mertens et al. (1993) Brit. J.Haematol. 85:133-42. Other methods known to those of ordinary skill inthe art can also be used to determine whether a given moiety has FactorVIII activity. Such methods are useful for determining the Factor VIIIactivity of both the moiety itself (and therefore can be used as a“Factor VIII moiety) as well as the corresponding polymer-moietyconjugate.

Nonlimiting examples of Factor VIII moieties include the following:Factor VIII; Factor VIIIa; Factor VIII:C; Factor VIII:vWF; B-domaindeleted Factor VIII (and other truncated versions of Factor VIII);hybrid proteins, such as those described in U.S. Pat. No. 6,158,888;glycosylated proteins having Factor VIII activity, such as thosedescribed in U.S. Patent Application Publication No. US2003/0077752; andpeptide mimetics having Factor VIII activity. Preferred truncated FactorVIII versions (encompassed by the term “B-domain deleted Factor VIII)corresponds to a protein having the amino acid sequence of human FactorVIII (SEQ ID NO:1) having a deletion corresponding to at least 581 aminoacids within the region between Arg⁷⁵⁹ and Ser¹⁷⁰⁹, more preferablywherein the deletion corresponds to one of the region between Pro¹⁰⁰⁰and Asp¹⁵⁸², the region between Thr⁷⁷⁸ and Pro¹⁶⁵⁹, and the regionbetween Thr⁷⁷⁸ and Glu¹⁶⁹⁴. Biologically active fragments, deletionvariants, substitution variants or addition variants of any of theforegoing that maintain at least some degree of Factor VIII activity canalso serve as a Factor VIII moiety.

The moiety having Factor VIII activity can advantageously be modified toinclude one or more amino acid residues such as, for example, lysine,cysteine and/or arginine, in order to provide facile attachment of thepolymer to an atom within the side chain of the amino acid. Techniquesfor adding amino acid residues are well known to those of ordinary skillin the art. Reference is made to J. March, Advanced Organic Chemistry:Reactions Mechanisms and Structure, 4th Ed. (New York:Wiley-Interscience, 1992).

The Factor VIII moiety can be obtained from blood-derived sources. Forexample, Factor VIII can be fractionated from human plasma usingprecipitation and centrifugation techniques known to those of ordinaryskill in the art. See, for example, Wickerhauser (1976) Transfusion16(4):345-350 and Slichter et al. (1976) Transfusion 16(6):616-626.Factor VIII can also be isolated from human granulocytes. See Szmitkoskiet al. (1977) Haematologia (Budap.) 11(1-2):177-187.

In addition, the Factor VIII moiety can also be obtained fromrecombinant methods. Briefly, recombinant methods involve constructingthe nucleic acid encoding the desired polypeptide or fragment, cloningthe nucleic acid into an expression vector, transforming a host cell(e.g., bacteria, yeast, or mammalian cell such as Chinese hamster ovarycell or baby hamster kidney cell), and expressing the nucleic acid toproduce the desired polypeptide or fragment. Methods for producing andexpressing recombinant polypeptides in vitro and in prokaryotic andeukaryotic host cells are known to those of ordinary skill in the art.See, for example, U.S. Pat. No. 4,868,122.

To facilitate identification and purification of the recombinantpolypeptide, nucleic acid sequences that encode for an epitope tag orother affinity binding sequence can be inserted or added in-frame withthe coding sequence, thereby producing a fusion protein comprised of thedesired polypeptide and a polypeptide suited for binding. Fusionproteins can be identified and purified by first running a mixturecontaining the fusion protein through an affinity column bearing bindingmoieties (e.g., antibodies) directed against the epitope tag or otherbinding sequence in the fusion proteins, thereby binding the fusionprotein within the column. Thereafter, the fusion protein can berecovered by washing the column with the appropriate solution (e.g.,acid) to release the bound fusion protein, The recombinant polypeptidecan also be identified and purified by lysing the host cells, separatingthe polypeptide, e.g., by size exclusion chromatography, and collectingthe polypeptide. These and other methods for identifying and purifyingrecombinant polypeptides are known to those of ordinary skill in theart.

The compositions of the invention can comprise a plurality ofconjugates, each conjugate comprised of the same Factor VIII moiety(i.e., within the entire composition, only one type of Factor VIIImoiety is found). In addition, the composition can comprise a pluralityof conjugates wherein any given conjugate is comprised of a moietyselected from the group consisting of two or more different Factor VIIImoieties (i.e., within the entire composition, two or more differentFactor VIII moieties are found). Optimally, however, substantially allof the plurality of conjugates in the composition (e.g., 85% or more ofthe plurality of conjugates in the composition) are each comprised ofthe same Factor VIII moiety.

Moreover, it is preferred that the composition containing the conjugatesis free or substantially free from albumin. It is also preferred thatthe composition is free or substantially free of proteins that do nothave Factor VIII activity. Thus, it is preferred that the composition is85%, more preferably 95%, and most preferably 99% free from albumin.Additionally, it is preferred that the composition is 85%, morepreferably 95%, and most preferably 99% free from any protein that doesnot have Factor VIII activity.

As previously discussed, each conjugate comprises a Factor VIII moietyattached to a water-soluble polymer. With respect to the water-solublepolymer, the water-soluble polymer is nonpeptidic, nontoxic,non-naturally occurring and biocompatible. With respect tobiocompatibility, a substance is considered biocompatible if thebeneficial effects associated with use of the substance alone or withanother substance (e.g., active agent such a Factor VIII moiety) inconnection with living tissues (e.g., administration to a patient)outweighs any deleterious effects as evaluated by a clinician, e.g., aphysician. With respect to non-immunogenicity, a substance is considerednonimmunogenic if the intended use of the substance in vivo does notproduce an undesired immune response (e.g., the formation of antibodies)or, if an immune response is produced, that such a response is notdeemed clinically significant or important as evaluated by a clinician.It is particularly preferred that the water-soluble polymer isbiocompatible and nonimmunogenic.

Further the polymer is typically characterized as having from 2 to about300 termini. Examples of such polymers include, but are not limited to,poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propyleneglycol) (“PPG”), copolymers of ethylene glycol and propylene glycol andthe like, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), and combinations of any of the foregoing.

The polymer is not limited in a particular structure and can be linear(e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g.,forked PEG or PEG attached to a polyol core), dendritic, or withdegradable linkages. Moreover, the internal structure of the polymer canbe organized in any of number of different patterns and can be selectedfrom the group consisting of homopolymer, alternating copolymer, randomcopolymer, block copolymer, alternating tripolymer, random tripolymer,and block tripolymer.

Typically, PEG and other water-soluble polymers are activated with asuitable activating group appropriate for coupling to a desired site onthe Factor VIII moiety. An activated polymeric reagent will possess areactive group for reaction with the Factor VIII moiety. Representativepolymeric reagents and methods for conjugating these polymers to anactive moiety are known in the art and further described in Zalipsky,S., et al., “Use of Functionalized Poly(Ethylene Glycols) forModification of Polypeptides” in Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press,New York (1992), and in Zalipsky (1995) Advanced Drug Reviews16:157-182.

Typically, the nominal average molecular weight of any given polymer inthe conjugate is from about 100 Daltons to about 150,000 Daltons.Exemplary ranges, however, include nominal average molecular weights inthe range of greater than 5,000 Daltons to about 100,000 Daltons, in therange of from about 6,000 Daltons to about 90,000 Daltons, in the rangeof from about 10,000 Daltons to about 85,000 Daltons, in the range offrom about 20,000 Daltons to about 85,000 Daltons, and in the range offrom about 53,000 Daltons to about 85,000 Daltons. For any givenwater-soluble polymer, PEGs having these molecular weight ranges arepreferred.

Exemplary nominal average molecular weights for the water-solublepolymer segment include about 100 Daltons, about 200 Daltons, about 300Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons,about 1,000 Daltons, about 2,000 Daltons, about 2,200 Daltons, about2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400Daltons, about 5,000 Daltons, about 6,000 Daltons, about 7,000 Daltons,about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000Daltons, about 40,000 Daltons, about 50,000 Daltons, about 60,000Daltons, and about 75,000 Daltons.

When used as the polymer, PEGs will typically comprise a number of(OCH₂CH₂) monomers. As used throughout the description, the number ofrepeating units is identified by the subscript “n” in “(OCH₂CH₂)_(n).”Thus, the value of (n) typically falls within one or more of thefollowing ranges: from 2 to about 2,300, from about 100 to about 2,300,from about 135 to about 2,000, from about 230 to about 1,900, from about450 to about 1,900, and from about 1,200 to about 1,900. For any givenpolymer in which the molecular weight is known, it is possible todetermine the number of repeating units (i.e., “n”) by dividing thetotal molecular weight of the polymer by the molecular weight of therepeating unit.

One particularly preferred polymer for use in the invention is anend-capped polymer, that is, a polymer having at least one terminuscapped with a relatively inert group, such as a lower C₁₋₆ alkoxy group.When the polymer is PEG, for example, it is preferred to use amethoxy-PEG (commonly referred to as mPEG), which is a linear form ofPEG wherein one terminus of the polymer is a methoxy (—OCH₃) group,while the other terminus is a hydroxyl or other functional group thatcan be optionally chemically modified.

In one form useful in the present invention, free or unbound PEG is alinear polymer terminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OH,wherein (m′) typically ranges from zero to about 4,000.

The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol can represent the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—,wherein (m′) is as defined as above.

Another type of PEG useful in the present invention is methoxy-PEG-OH,or mPEG in brief, in which one terminus is the relatively inert methoxygroup, while the other terminus is a hydroxyl group. The structure ofmPEG is given below.CH₃O—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OHwherein (m′) is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, can also be used as the PEG polymer. For example,PEG can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones (either the same or different),such as methoxy poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine. Depending on the specific Factor VIII moiety used, the reactiveester functional group of the disubstituted lysine may be furthermodified to form a functional group suitable for reaction with thetarget group within the Factor VIII moiety.

These polymers may be linear, or may be in any of the above-describedforms.

In addition, the PEG can comprise a forked PEG. An example of a forkedPEG is represented by the following structure:

wherein: X is a spacer moiety of one or more atoms and each Z is anactivated terminal group linked to CH by a chain of atoms of definedlength. International Application No. PCT/US99/05333, discloses variousforked PEG structures capable of use in the present invention. The chainof atoms linking the Z functional groups to the branching carbon atomserve as a tethering group and may comprise, for example, alkyl chains,ether chains, ester chains, amide chains and combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG rather than at the end of the PEG chain. The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymer,including any of the above described polymers. For example, PEG can beprepared with ester linkages in the polymer that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include: carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); phosphateester linkages formed, for example, by reacting an alcohol with aphosphate group; hydrazone linkages which are typically formed byreaction of a hydrazide and an aldehyde; acetal linkages that aretypically formed by reaction between an aldehyde and an alcohol;orthoester linkages that are, for example, formed by reaction between aformate and an alcohol; amide linkages formed by an amine group, e.g.,at an end of a polymer such as PEG, and a carboxyl group of another PEGchain; urethane linkages formed from reaction of, e.g., a PEG with aterminal isocyanate group and a PEG alcohol; peptide linkages formed byan amine group, e.g., at an end of a polymer such as PEG, and a carboxylgroup of a peptide; and oligonucleotide linkages formed by, for example,a phosphoramidite group, e.g., at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.

Such optional features of the polymer conjugate, i.e., the introductionof one or more degradable linkages into the polymer chain, may providefor additional control over the final desired pharmacological propertiesof the conjugate upon administration. For example, a large andrelatively inert conjugate (i.e., having one or more high molecularweight PEG chains attached thereto, for example, one or more PEG chainshaving a molecular weight greater than about 10,000, wherein theconjugate possesses essentially no bioactivity) may be administered,which is hydrolyzed to generate a bioactive conjugate possessing aportion of the original PEG chain. In this way, the properties of theconjugate can be more effectively tailored to balance the bioactivity ofthe conjugate over time.

Those of ordinary skill in the art will recognize that the foregoingdiscussion concerning substantially water-soluble polymer segments is byno means exhaustive and is merely illustrative, and that all polymericmaterials having the qualities described above are contemplated. As usedherein, the term “polymeric reagent” generally refers to an entiremolecule, which can comprise a water-soluble polymer segment and afunctional group.

As described above, a conjugate of the invention comprises awater-soluble polymer covalently attached to a Factor VIII moiety.Typically, for any given conjugate, there will be one to threewater-soluble polymers covalently attached to one or more moietieshaving Factor VIII activity. In some instances, however, the conjugatemay have 1, 2, 3, 4, 5, 6, 7, 8 or more water-soluble polymersindividually attached to a Factor VIII moiety.

The particular linkage within the moiety having Factor VIII activity andthe polymer depends on a number of factors. Such factors include, forexample, the particular linkage chemistry employed, the particularmoiety having Factor VIII activity, the available functional groupswithin the moiety having Factor VIII activity (either for attachment toa polymer or conversion to a suitable attachment site), the possiblepresence of additional reactive functional groups within the moietyhaving Factor VIII activity, and the like.

The conjugates of the invention can be, although not necessarily,prodrugs, meaning that the linkage between the polymer and the FactorVIII moiety is hydrolytically degradable to allow release of the parentmoiety. Exemplary degradable linkages include carboxylate ester,phosphate ester, thiolester, anhydrides, acetals, ketals, acyloxyalkylether, imines, orthoesters, peptides and oligonucleotides. Such linkagescan be readily prepared by appropriate modification of either the FactorVIII moiety (e.g., the carboxyl group C terminus of the protein or aside chain hydroxyl group of an amino acid such as serine or threoninecontained within the protein) and/or the polymeric reagent usingcoupling methods commonly employed in the art. Most preferred, however,are hydrolyzable linkages that are readily formed by reaction of asuitably activated polymer with a non-modified functional groupcontained within the moiety having Factor VIII activity.

Alternatively, a hydrolytically stable linkage, such as an amide,urethane (also known as carbamate), amine, thioether (also known assulfide), or urea (also known as carbamide) linkage can also be employedas the linkage for coupling the Factor VIII moiety. In some cases,however, it is preferred that the linkage is not a carbamate linkage andnot a carbamide linkage, and furthermore, that no linkage is formedbased on the reaction of a polymer derivative bearing an isocyanate orisothiocyanate species to a Factor VIII moiety. Again, a preferredhydrolytically stable linkage is an amide. An amide can be readilyprepared by reaction of a carboxyl group contained within the FactorVIII moiety (e.g., the terminal carboxyl of a peptidic moiety havingFactor VIII activity) with an amino-terminated polymer.

The conjugates (as opposed to an unconjugated Factor VIII moiety) may ormay not possess a measurable degree of Factor VIII activity. That is tosay, a polymer conjugate in accordance with the invention will possessesanywhere from about 0.1% to about 100% or more of the bioactivity of theunmodified parent Factor VIII moiety. Preferably, compounds possessinglittle or no Factor VIII activity typically contain a hydrolyzablelinkage connecting the polymer to the moiety, so that regardless of thelack of activity in the conjugate, the active parent molecule (or aderivative thereof) is released upon aqueous-induced cleavage of thehydrolyzable linkage. Such activity may be determined using a suitablein-vivo or in-vitro model, depending upon the known activity of theparticular moiety having Factor VIII activity employed.

For conjugates possessing a hydrolytically stable linkage that couplesthe moiety having Factor VIII activity to the polymer, the conjugatewill typically possess a measurable degree of specific activity. Forinstance, such polymer conjugates are typically characterized as havingan activity of at least about 2%, 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%,80%, 85%, 90%, 95% 97%, 100%, or more relative to that of the unmodifiedparent moiety having Factor VIII activity, when measured in a suitablemodel, such as those well known in the art. Preferably, compounds havinga hydrolytically stable linkage (e.g., an amide linkage) will possess atleast some degree of the bioactivity of the unmodified parent moietyhaving Factor VIII activity.

Exemplary polymer conjugates in accordance with the invention will nowbe described wherein the moiety having Factor VIII activity is aprotein. Typically, such a protein is expected to share (at least inpart) a similar amino acid sequence as native Factor VIII. Thus, whilereference will be made to specific locations or atoms within the nativeFactor VIII protein, such a reference is for convenience only and onehaving ordinary skill in the art will be able to readily determine thecorresponding location or atom in other moieties having Factor VIIIactivity. In particular, the description provided herein for nativeFactor VIII is applicable to Factor VIIIa, Factor VIII:vWF, and B-domaindeleted Factor VIII versions, as well as fragments, deletion variants,substitution variants or addition variants of any of the foregoing.

Amino groups on Factor VIII moieties provide a point of attachmentbetween the Factor VIII moiety and the water-soluble polymer. NativeFactor VIII comprises 158 amine-containing lysine residues (6.8 weightpercent of the entire protein) and one amino terminus. With respect toFactor VIIIa, there are 78 lysine residues (5.5 weight percent of theentire protein) and two amino termini (resulting from the cleavage ofFactor VIII). Consequently, notwithstanding secondary and tertiarystructural considerations, both Factor VIII and Factor VIIIa (as well asany peptidic Factor VIII moiety, e.g., B-domain deleted Factor VIII)have several amines available for participation in conjugatingreactions.

There are a number of examples of suitable water-soluble polymericreagents useful for forming covalent linkages with available amines of aFactor VIII moiety. Specific examples, along with the correspondingconjugate, are provided in Table 1, below. In the table, the variable(n) represents the number of repeating monomeric units and “—NH—F8”represents the Factor VIII moiety following conjugation to thewater-soluble polymer. While each polymeric portion presented in Table 1terminates in a “CH₃” group, other groups (such as H and benzyl) can besubstituted therefor.

TABLE 1 Amine-Specific Polymeric Reagents and the Factor VIII MoietyConjugate Formed Therefrom Polymeric Reagent

mPEG-Succinimidyl Propionate

Homobifunctional PEG-Succinimidyl Propionate

mPEG-Succinimidyl Butanoate

mPEG-Benzotriazole Carbonate

mPEG-Succinimidyl Derivative

Branched mPEG2-N-Hydroxysuccinimide

mPEG-Succinimidyl Derivative

mPEG-Succinimidyl Derivative

Homobifunctional PEG Propionaldehyde

mPEG Propionaldehyde

Homobifunctional PEG Butyraldehyde

mPEG Butryaldehyde

mPEG Butryaldehyde Derivative

Branched mPEG2 Butyraldehyde

mPEG Acetal

mPEG Piperidone

mPEG Methylketone

mPEG “Linkerless” Maleimide (under certain reaction conditions such aspH > 8)

mPEG Maleimide Derivative (under certain reaction conditions such aspH > 8)

mPEG Maleimide Derivative (under certain reaction conditions such aspH > 8)

mPEG Forked Maleimide Derivative (under certain reaction conditions suchas pH > 8)

branched mPEG2 Maleimide Derivative (under certain reaction conditionssuch as pH > 8) Corresponding Conjugate

Amide Linkage

Amide Linkages

Amide Linkage

Carbamate Linkage

Carbamate Linkage

Amide Linkage

Amide Linkage

Amide Linkage

Amide Linkage

Secondary Amine Linkages

Secondary Amine Linkage

Secondary Amine Linkage

Secondary Amine Linkage

Secondary Amine Linkage

Secondary Amine Linkage

Secondary Amine Linkage

Secondary Amine Linkage (to a secondary carbon)

Secondary amine Linkage (to a secondary carbon)

Secondary Amine Linkage

Secondary Amine Linkage

Secondary Amine Linkage

Secondary Amine Linkages

Secondary Amine Linkage

Conjugation of a polymeric reagent to an amino group of a Factor VIIImoiety can be accomplished by one of ordinary skill in the art withoutundue experimentation. Typical of one approach is a reductive aminationreaction used, for example, to conjugate a primary amine of a FactorVIII moiety with a polymer functionalized with a ketone, aldehyde orhydrated forms thereof (e.g., ketone hydrate, aldehyde hydrate). In thisapproach, the primary amine from the Factor VIII moiety reacts with thecarbonyl group of the aldehyde or ketone (or the correspondinghydroxy-containing group of a hydrated aldehyde or ketone), therebyforming a Schiff base. The Schiff base, in turn, can then be reductivelyconverted to a stable conjugate through use of a reducing agent such assodium borohydride. Selective reactions (e.g., at the N-terminus arepossible) are possible, particularly with a polymer functionalized witha ketone or an alpha-methyl branched aldehyde and/or under specificreaction conditions (e.g., reduced pH).

Preferred amine groups in Factor VIII that can serve as a site forattaching a polymer include those amine groups found within thefollowing lysine residues: Lys⁴⁹³, Lys⁴⁹⁶, Lys⁴⁹⁹, Lys¹⁸⁰⁴, Lys¹⁸⁰⁸,Lys¹⁸¹³, Lys¹⁸¹⁸, Lys²¹⁸³, Lys²²⁰⁷, Lys²²²⁷, Lys²²³⁶, with Lys⁴⁹⁶,Lys¹⁸⁰⁴, and Lys¹⁸⁰⁸ being particularly preferred. Numbering correspondsto the sequence provided in SEQ ID NO:2. As stated above, the aminegroup corresponding to each of these lysine residues in a protein otherthan human Factor VIII can serve as a useful site for conjugation. Inaddition, the N-terminus of any Factor VIII moiety that is a protein canserve as a polymeric attachment site.

Carboxyl groups represent another functional group that can serve as apoint of attachment on the Factor VIII moiety. Structurally, theconjugate will comprise the following:

where F8 and the adjacent carbonyl group corresponds to thecarboxyl-containing Factor VIII moiety, X is a linkage, preferably aheteroatom selected from O, N(H), and S, and POLY is a water-solublepolymer such as PEG, optionally terminating in an end-capping moiety.

The C(O)—X linkage results from the reaction between a polymericderivative bearing a terminal functional group and a carboxyl-containingFactor VIII moiety. As discussed above, the specific linkage will dependon the type of functional group utilized. If the polymer isend-functionalized or “activated” with a hydroxyl group, the resultinglinkage will be a carboxylic acid ester and X will be O. If the polymerbackbone is functionalized with a thiol group, the resulting linkagewill be a thioester and X will be S. When certain multi-arm, branched orforked polymers are employed, the C(O)X moiety, and in particular the Xmoiety, may be relatively more complex and may include a longer linkagestructure.

Water-soluble derivatives containing a hydrazide moiety are also usefulfor conjugation at carboxyl groups. An example of such a derivativeincludes a polymer having the following structure:

which, upon conjugation with a Factor VIII moiety, has the followingstructure:

where F8 is the Factor VIII moiety following conjugation and POLY is awater-soluble polymer such as PEG, optionally terminating in anend-capping moiety.

Thiol groups contained within the Factor VIII moiety can serve aseffective sites of attachment for the water-soluble polymer. Inparticular, cysteine residues provide thiol groups when the Factor VIIImoiety is a protein. The thiol groups in such cysteine residues can thenbe reacted with an activated PEG that is specific for reaction withthiol groups, e.g., an N-maleimidyl polymer or other derivative, asdescribed in U.S. Pat. No. 5,739,208 and in International PatentPublication No. WO 01/62827.

Specific examples, along with the corresponding conjugate, are providedin Table 2, below. In the table, the variable (n) represents the numberof repeating monomeric units and “—S—F8” represents the Factor VIIImoiety following conjugation to the water-soluble polymer. While eachpolymeric portion presented in Table 1 terminates in a “CH₃” group,other groups (such as H and benzyl) can be substituted therefor.

TABLE 2 Thiol-Specific Polymeric Reagents and the Factor VIII MoietyConjugate Formed Therefrom Polymeric Reagent

mPEG “Linkerless” Maleimide

mPEG Maleimide Derivative

mPEG Maleimide Derivative

mPEG Forked Maleimide Derivative

branched mPEG2 Maleimide Derivative

Branched mPEG2 Forked Maleimide Derivative

mPEG vinyl sulfone

mPEG thiol Corresponding Conjugate

Thioether Linkage

Thioether Linkage

Thioether Linkage

Thioether Linkage

Thioether Linkage

Thioether Linkages

Thioether Linkage

Disulfide Linkage

With respect to conjugates formed from water-soluble polymers bearingone or more maleimide functional groups (regardless of whether themaleimide reacts with an amine or thiol group on the Factor VIIImoiety), the corresponding maleamic acid form(s) of the water-solublepolymer can also react with the Factor VIII moiety. Under certainconditions (e.g., a pH of about 7-9 and in the presence of water), themaleimide ring will “open” to form the corresponding maleamic acid. Themaleamic acid, in turn, can react with an amine or thiol group of aFactor VIII moiety. Exemplary maleamic acid-based reactions areschematically shown below. POLY represents the water-soluble polymer,and F8 represents the Factor VIII moiety.

A representative conjugate in accordance with the invention can have thefollowing structure:POLY-L_(0,1)-C(O)Z—Y—S—S—F8wherein POLY is a water-soluble polymer, L is an optional linker, Z is aheteroatom selected from the group consisting of O, NH, and S, and Y isselected from the group consisting of C₂₋₁₀ alkyl, C₂₋₁₀ substitutedalkyl, aryl, and substituted aryl. Polymeric reagents that can bereacted with a Factor VIII moiety and result in this type of conjugateare described in copending application filed on Jan. 6, 2004, entitled“Thiol Selective Water Soluble Polymer Derivatives,” and assigned U.S.Ser. No. 10/753,047.

Preferred thiol groups in Factor VIII that can serve as a site forattaching a polymeric reagent include those thiol groups found withinthe following cysteine residues: Cys²⁴⁸, Cys³¹⁰, Cys³²⁹, Cys⁶³⁰, Cys⁶⁹²,Cys⁷¹¹, Cys¹⁸⁹⁹, Cys¹⁹⁰³, and Cys²⁰⁰⁰, with Cys⁶³⁰, Cys⁷¹¹, and Cys¹⁹⁰³,being particularly preferred. Numbering corresponds to the sequenceprovided in SEQ ID NO:2.

With respect to polymeric reagents, those described here and elsewherecan be purchased from commercial sources (e.g., Nektar Therapeutics,Huntsville Ala.). In addition, methods for preparing the polymericreagents are described in the literature.

Typically, although not necessarily, the linkage between the Factor VIIImoiety and the polymeric reagent includes one or more atoms such as oneor more of carbon, nitrogen, sulfur, and combinations thereof.Preferably, the linkage comprises an amide, secondary amine, carbamate,thioether, or disulfide group. Optionally, additional atoms can connectthe linkage to the chain of repeating monomers within the polymericreagent. Nonlimiting examples of specific series of atoms connecting theFactor VIII moiety to the chain of repeating monomers include thoseselected from the group consisting of —O—, —S—, —S—S—, —C(O)—, —O—C(O)—,—C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—,—CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—,—O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—,—CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]₀₋₆—(OCH₂CH₂)₀₋₂—, —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —O—C(O)—CH₂—, —O—C(O)—CH₂—CH₂—, and—O—C(O)—CH₂—CH₂—CH₂—.

The conjugates are typically part of a composition. Generally, thecomposition comprises a plurality of conjugates, preferably although notnecessarily, each having one to three water-soluble polymers covalentlyattached to one Factor VIII moiety. The compositions, however, can alsocomprise other conjugates having four, five, six, seven, eight or morepolymers attached to any given moiety having Factor VIII activity. Inaddition, the invention includes instances wherein the compositioncomprises a plurality of conjugates, each conjugate comprising onewater-soluble polymer covalently attached to one Factor VIII moiety, aswell as compositions comprising two, three, four, five, six, seven,eight, or more water-soluble polymers covalently attached to one FactorVIII moiety.

Control of the desired number of polymers for any given moiety can beachieved by selecting the proper polymeric reagent, the ratio ofpolymeric reagent to the Factor VIII moiety, temperature, pH conditions,and other aspects of the conjugation reaction. In addition, reduction orelimination of the undesired conjugates (e.g., those conjugates havingfour or more attached polymers) can be achieved through purificationmeans.

For example, the polymer-Factor VIII moiety conjugates can be purifiedto obtain/isolate different conjugated species. Specifically, theproduct mixture can be purified to obtain an average of anywhere fromone, two, three, four, five or more PEGs per Factor VIII moiety,typically one, two or three PEGs per Factor VIII moiety. The strategyfor purification of the final conjugate reaction mixture will dependupon a number of factors, including, for example, the molecular weightof the polymeric reagent employed, the particular Factor VIII moiety,the desired dosing regimen, and the residual activity and in vivoproperties of the individual conjugate(s).

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography and/or ion exchangechromatography. That is to say, gel filtration chromatography is used tofractionate differently numbered polymer-to-Factor VIII moiety ratios(e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates 1polymer to Factor VIII moiety, “2-mer” indicates two polymers to FactorVIII moiety, and so on) on the basis of their differing molecularweights (where the difference corresponds essentially to the averagemolecular weight of the water-soluble polymer portion). For example, inan exemplary reaction where a 100,000 Dalton protein is randomlyconjugated to a polymeric reagent having a molecular weight of about20,000 Daltons, the resulting reaction mixture may contain unmodifiedprotein (having a molecular weight of about 100,000 Daltons),monoPEGylated protein (having a molecular weight of about 120,000Daltons), diPEGylated protein (having a molecular weight of about140,000 Daltons), and so forth.

While this approach can be used to separate PEG and other polymer-FactorVIII moiety conjugates having different molecular weights, this approachis generally ineffective for separating positional isomers havingdifferent polymer attachment sites within the Factor VIII moiety. Forexample, gel filtration chromatography can be used to separate from eachother mixtures of PEG 1-mers, 2-mers, 3-mers, and so forth, althougheach of the recovered PEG-mer compositions may contain PEGs attached todifferent reactive amino groups (e.g., lysine residues) within FactorVIII moiety.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, (iii) iodine testing for PEG content (Sims et al. (1980) Anal.Biochem, 107:60-63), and (iv) sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS PAGE), followed by staining with barium iodide.

Separation of positional isomers is carried out by reverse phasechromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) orby ion exchange chromatography using an ion exchange column, e.g., aSepharose™ ion exchange column available from Amersham Biosciences.Either approach can be used to separate polymer-active agent isomershaving the same molecular weight (positional isomers).

The compositions are preferably substantially free of proteins that donot have Factor VIII activity. In addition, the compositions preferablyare substantially free of all other noncovalently attached water-solublepolymers. Moreover, at least one species of conjugate in the compositionhas at least one water-soluble water polymer attached to a moiety thattransforms Factor X to Factor Xa. In some circumstances, however, thecomposition can contain a mixture of polymer-Factor VIII moietyconjugates and unconjugated Factor VIII.

Optionally, the composition of the invention further comprises apharmaceutically acceptable excipient. If desired, the pharmaceuticallyacceptable excipient can be added to a conjugate to form a composition.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The composition can also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the composition as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfate, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant can be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as TWEEN 20™ (Polysorbate 20) and TWEEN® 80(Polysorbate 80) and pluronics such as PLURONIC® F68(polyoxyethylene-polyoxypropylene block copolymer) and PLURONIC® F88(both of which are available from BASF, Mount Olive, N.J.); sorbitanesters; lipids, such as phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines (although preferably notin liposomal form), fatty acids and fatty esters; steroids, such ascholesterol; and chelating agents, such as EDTA, zinc and other suchsuitable cations.

Acids or bases can be present as an excipient in the composition.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The amount of the conjugate (i.e., the conjugate formed between theactive agent and the polymeric reagent) in the composition will varydepending on a number of actors, but will optimally be a therapeuticallyeffective dose when the composition is stored in a unit dose container(e.g., a vial). In addition, the pharmaceutical preparation can behoused in a syringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about 5%to about 98% by weight, more preferably from about 15 to about 95% byweight of the excipient, with concentrations less than 30% by weightmost preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The compositions encompass all types of formulations and in particularthose that are suited for injection, e.g., powders or lyophilates thatcan be reconstituted as well as liquids. Examples of suitable diluentsfor reconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof. With respect to liquidpharmaceutical compositions, solutions and suspensions are envisioned.

The compositions of the present invention are typically, although notnecessarily, administered via injection and are therefore generallyliquid solutions or suspensions immediately prior to administration. Thepharmaceutical preparation can also take other forms such as syrups,creams, ointments, tablets, powders, and the like. Other modes ofadministration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalcomposition). As previously described, the conjugates can beadministered injected parenterally by intravenous injection, or lesspreferably by intramuscular or by subcutaneous injection. Suitableformulation types for parenteral administration includeready-for-injection solutions, dry powders for combination with asolvent prior to use, suspensions ready for injection, dry insolublecompositions for combination with a vehicle prior to use, and emulsionsand liquid concentrates for dilution prior to administration, amongothers.

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the conjugate. Those ofordinary skill in the art appreciate which conditions a specificconjugate can effectively treat. For example, the conjugates can be usedto treat individuals suffering from hemophilia A. In addition, theconjugates are suited for use as a prophylactic against uncontrolledbleeding, optionally in patients not suffering from hemophilia. Thus,for example, the conjugate can be administered to a patient at risk foruncontrolled bleeding prior to surgery.

The actual dose to be administered will vary depend upon the age,weight, and general condition of the subject as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and conjugate being administered. Therapeuticallyeffective amounts are known to those skilled in the art and/or aredescribed in the pertinent reference texts and literature. Generally, atherapeutically effective amount will range from about 0.001 mg to 100mg, preferably in doses from 0.01 mg/day to 75 mg/day, and morepreferably in doses from 0.10 mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering certain conjugates of the presentinvention is that individual water-soluble polymer portions can becleaved off. Such a result is advantageous when clearance from the bodyis potentially a problem because of the polymer size. Optimally,cleavage of each water-soluble polymer portion is facilitated throughthe use of physiologically cleavable and/or enzymatically degradablelinkages such as urethane, amide, carbonate or ester-containinglinkages. In this way, clearance of the conjugate (via cleavage ofindividual water-soluble polymer portions) can be modulated by selectingthe polymer molecular size and the type functional group that wouldprovide the desired clearance properties. One of ordinary skill in theart can determine the proper molecular size of the polymer as well asthe cleavable functional group. For example, one of ordinary skill inthe art, using routine experimentation, can determine a proper molecularsize and cleavable functional group by first preparing a variety ofpolymer derivatives with different polymer weights and cleavablefunctional groups, and then obtaining the clearance profile (e.g.,through periodic blood or urine sampling) by administering the polymerderivative to a patient and taking periodic blood and/or urine sampling.Once a series of clearance profiles have been obtained for each testedconjugate, a suitable conjugate can be identified.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis and the like, which arewithin the skill of the art. Such techniques are fully explained in theliterature. See, for example, J. March, Advanced Organic Chemistry:Reactions Mechanisms and Structure, 4th Ed. (New York:Wiley-Interscience, 1992), supra.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric pressure at sea level.

ABBREVIATIONS:

DCM dichloromethane

mPEG-SPA mPEG-succinimidyl propionate

mPEG-SBA mPEG-succinimidyl butanoate

mPEG-OPSS mPEG-orthopyridyl-disulfide

mPEG-MAL mPEG-maleimide, CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂-MAL

mPEG-SMB mPEG-succinimidyl α-methylbutanoate,CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—CH(CH₃)—C(O)—O-succinimide

mPEG-ButyrALDCH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—O—C(O)—NH—(CH₂CH₂(O)₄CH₂CH₂CH₂C(O)H

mPEG-PIP CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)-piperidin-4-one

SUC succinimide or succinimidyl

NaCNBH₃ sodium cyanoborohydride

HCl hydrochloric acid

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

NMR nuclear magnetic resonance

DCC 1,3-dicyclohexylcarbodiimide

DI deionized

MW molecular weight

r.t. room temperature

K or kDa kilodaltons

SEC Size exclusion chromatography

HPLC high performance liquid chromatography

FPLC fast protein liquid chromatography

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

MALDI-TOF Matrix Assisted Laser Desorption Ionization Time-of-Flight

Materials:

All PEG reagents referred to in the appended examples are commerciallyavailable unless otherwise indicated.

mPEG-succinimidyl propionate, mPEG-SPA, molecular weight, 30K(M_(n)=31.3 kDa, Nektar Therapeutics)

mPEG-orthopyridyl-disulfide, mPEG-OPSS, molecular weight, 10K(M_(n)=10.3 kDa, Nektar Therapeutics)

mPEG-maleimide, mPEG-MAL, molecular weight, 20K (Mn=21.8 kDa, NektarTherapeutics)

mPEG-maleimide, mPEG-MAL, molecular weight, 30K (M_(n)31.4 kDa, NektarTherapeutics)

mPEG-succinimidyl α-methylbutanoate, mPEG-SMB, molecular weight, 30K(M_(n)=30.5 kDa, Nektar Therapeutics)

mPEG-butyraldehyde, mPEG-ButyrALD, molecular weight, 30K (M_(n)=31.5kDa, Nektar Therapeutics)

L-Histidine, biotechnology performance certified (Sigma)

HEPES, biotechnology performance certified, 99.5+% (Sigma)

Calcium chloride, dihydrate, for molecular biology, 99% (Sigma)

Sodium chloride, for molecular biology (Sigma)

TWEEN 80™ (Polyoxyethylene 20 sorbitan monooleate), Sigma Ultra,(Sigma).

Ethyl alcohol, USP, Absolute-200 Proof (AAPER)

Polyethylene glycol, MW 3,350, SigmaUltra (Sigma)

Slide-A-Lyzer Dialysis Cassette, 0.5-3 ml, or 3-12 ml capacity (Pierce)

Acetic acid, A.C.S. reagent, 99.7+% (Aldrich)

1N Acetic acid, volumetric standard (VWR)

1N Sodium hydroxide, volumetric standard (J. T. Baker)

Sodium cyanoborohydride, 95% (Aldrich)

Tris/glycine/SDS, 10×, protein electrophoresis buffer (Bio-Rad)

Laemmli sample buffer (Bio-Rad)

SigmaMarker, low range (M.W. 6,500-66,000) (Sigma)

SigmaMarker, high range (M.W. 36,000-205,000) (Sigma)

7.5% Tris-HCl ready gel (10-well, 30 ul, Bio-Rad)

GelCode Blue Stain Reagent (Pierce)

Methods (Analytical)

SEC-HPLC Analysis

Size exclusion chromatography (SEC) was performed on an Agilent 1100HPLC system (Agilent). Samples were analyzed using a BIOSEP-SEC-S 4000column (Phenomenex) and a mobile phase of 45 mM histidine, 4.5 mMcalcium chloride, 0.36 M sodium chloride, 0.009%(v/v) Tween 80 and 10%ethyl alcohol, pH 6.7. The flow rate for the column was 0.3 ml/min.Eluted protein and PEG-protein conjugates were detected by UV at awavelength of 280 nm.

SDS-PAGE Analysis

Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) using Mini-PROTEAN 3 Precast GelElectrophoresis System (Bio-Rad). Samples were mixed with 2× Laemmlisample buffer, and were placed in a 95° C. water bath for ˜5 minutes.Then, the prepared samples were loaded onto a 7.5% Tris-HCl ready geland run for approximately 30 minutes at 200 V using Tris/glycine/SDSelectrophoresis buffer.

Other Methods

Purification of PEG-Factor VIII Conjugates

A Superose 6 HR 10/30, 24 ml gel filtration column (Amersham) was usedwith an FPLC system and AKTA prime system (Amersham) to purify thePEG-Factor VIII conjugates in Examples 6-11. The flow rate was 0.3ml/min and the elution buffer was 50 mM Histidine, 0.5 M NaCl, 4.0 mMCaCl₂, and 0.01% (w/v) TWEEN 80™ (Polyoxyethylene 20 sorbitanmonooleate), pH 6.7.

Buffer Exchange of Factor VIII Stock Solution

A Slide-A-Lyzer Dialysis Cassette (3-12 ml, 10,000 MWCO, Pierce) wasremoved from the protective pouch, and was soaked in MiliQ H₂O for 15minutes (water was changed every 5 minutes). The Factor VIII stocksolution [0.398 mg/ml in 50 mM Histidine, 0.5 M NaCl, 4.0 mM CaCl₂, 0.1%(w/v) PEG 3,350, 0.01% (w/v) TWEEN 80™ (Polyoxyethylene 20 sorbitanmonooleate), pH 6.7] was then transferred into the cassette cavitythrough one of the guide ports on the top of the gasket. The cassettewas placed in a 1 L beaker of HEPES buffer [50 mM HEPES, 0.5 M NaCl, 5mM CaCl₂, 0.1% (w/v) PEG 3,350, 0.01% (v/v) TWEEN 80™ (Polyoxyethylene20 sorbitan monooleate), pH 7.0] with a flotation buoy attached to thetop of the cassette. The beaker was then placed on the stir plate tostart the dialysis at 4° C. The HEPES buffer was changed for four timesat intervals of 2-3 hrs, and was then left in a cold room (4° C.) forovernight dialysis. After dialysis, the cassette chamber was injectedwith air and the dialyzed sample was withdrawn from the cassette. Theconcentration of Factor VIII in HEPES buffer was measured at UV 280 nmfrom a SPECTRA max PLUS Spectrophotometer (Molecular Devices).

Example 1 PEGylation of B-Domain Deleted Factor VIII with mPEG-SPA, 20K

mPEG-Succinimidyl propionate having a molecular weight of 20,000 Daltonsis obtained from Nektar Therapeutics, (Huntsville, Ala.). The basicstructure of the polymer reagent is provided below:

B-domain deleted Factor VIII is dissolved in deionized water, to whichis added triethylamine to raise the pH to 7.2-9. To the above solutionis added a 1.5 to 10-fold molar excess of the PEG reagent, mPEG-SPA. Theresulting mixture is stirred at room temperature for several hours.

The reaction mixture is analyzed by SDS-PAGE to determine the degree ofPEGylation of the protein. The degree of PEGylation, 1-mer, 2 mers,etc., can also be determined by any of a number of analytical techniquesappropriate for proteins of this size, such as light angle scattering.The displayed peaks for native and mono-PEGylated species differ byapproximately 20,000 Da. Increasing the ratio of PEG reagent to proteinincreases the degree of polyPEGylation, that is to say, the formation of2-mers, 3-mers, etc.

Example 2 PEGylation of B-Domain Deleted Factor VIII with mPEG-SBA

mPEG-Succinimidyl butanoate having a molecular weight of 10,000 daltonsis obtained from Nektar Therapeutics, (Huntsville, Ala.). The basicstructure of the polymer reagent is provided below:

B-domain deleted Factor VIII is dissolved in deionized water, to whichis added triethylamine to raise the pH to 7.2-9. To this solution isthen added a 1.5 to 10-fold molar excess of mPEG-SBA. The resultingmixture is stirred at room temperature for several hours.

The reaction mixture is analyzed by SDS-PAGE to determine the degree ofPEGylation of the protein.

Example 3 PEGylation of B-Domain Deleted Factor VIII with mPEG-MAL, 20K

mPEG-Maleimide having a molecular weight of 20,000 daltons is obtainedfrom Nektar Therapeutics, (Huntsville, Ala.). The basic structure of thepolymer reagent is provided below:

B-domain deleted Factor VIII is dissolved in buffer. To this proteinsolution is added a 3-5 fold molar excess of mPEG-MAL. The mixture isstirred at room temperature under an inert atmosphere for several hours.The reaction mixture is analyzed and purified by HPLC to provide amixture of conjugated species.

Example 4 PEGylation of B-Domain Deleted Factor VIII with mPEG-OPSS, 20K

The sulfhydryl-selective polymer reagent, mPEG-orthopyridyl-disulfide(structure shown above), having a molecular weight of 20,000, isobtained from Nektar Therapeutics (Huntsville, Ala.). A five-fold molarexcess of mPEG-OPSS is added to B-domain deleted Factor VIII in abuffered solution. The reaction mixture is stirred for several hours atroom temperature under an inert atmosphere to form the desired conjugatehaving a disulfide linkage connecting the polymer to the protein.

Example 5 PEGylation of B-Domain Deleted Factor VIII with mPEG-PIP, 5K

The above polymeric reagent, shown as both the ketone and correspondingketal, is prepared as described in Nektar Therapeutics' ProvisionalPatent Application No. 60/437,325, entitled “Polymer Derivatives andConjugates Thereof.”

To prepare the above polymeric reagent, to a solution ofmethoxy-polyethylene glycol-succinimidyl propionate having a weightaverage molecular weight of 5,000 Daltons (1.0 g, 0.002 moles) inmethylene chloride (20 ml), triethyl amine (0.084 ml, 0.006 moles) and4-piperidone monohydrate hydrochloride (0.077 g, 0.005 moles) are added.The reaction mixture is stirred at room temperature under a nitrogenatmosphere overnight and then purified prior to conjugation.Alternatively, the polymer reagent may be purchased from NektarTherapeutics.

To effect conjugation, to a solution of B-domain deleted Factor VIII inaqueous buffer is added a 20-fold molar excess of mPEG-PIP, 5K. Theresulting solution is placed on a Roto Mix™ orbital shaker (ThermolyneCorp., Dubuque, Iowa) set at slow speed to facilitate reaction at roomtemperature. After 15 minutes, aqueous NaCNBH3 is added in an amountequal to a 50 fold-molar excess relative to the B-domain deleted FactorVIII. Aliquots are withdrawn at timed intervals from the reactionmixture and are analyzed by SDS-PAGE (using gels available from Bio-RadLaboratories, Hercules, Calif.).

SDS-PAGE analysis indicates the presence of PEG derivatives of B-domaindeleted Factor VIII having 1, 2, and 3 PEG moieties attached.

Example 6 Conjugation of B-Domain Deleted Factor VIII with mPEG-SPA, 30K

Prior to conjugation, a buffer exchange for B-domain deleted Factor VIII(Factor VIII) was performed to replace histidine with HEPES.

mPEG-SPA, 30K, stored at −20° C. under argon, was warmed to ambienttemperature. The warmed mPEG-SPA (2.2 mg) was dissolved in 0.022 ml of 2mM HCl to provide a 10% solution of polymer reagent. The mPEG-SPAsolution was quickly added to 3 ml of Factor VIII solution [0.412 mg/mlin 50 mM HEPES, 0.5 M NaCl, 4.0 mM CaCl₂, 0.1% (w/v) PEG 3,350, 0.01%(w/v) TWEEN 80™ (Polyoxyethylene 20 sorbitan monooleate), pH 7.0] andmixed well. After 30 minutes of reaction at room temperature, thereaction vial was transferred to a cold room (4° C.), and another 0.022ml of mPEG-SPA solution was added to the reaction mixture, and mixedwell. The pH was determined (pH 7.0±0.2). The molar ratio of mPEG-SPA toprotein was 20:1. The final mPEG-SPA concentration was 1.445 mg/ml, andthe final Factor VIII concentration was 0.406 mg/ml. The reaction wasallowed to proceed overnight at 4° C. on Rotomix (slow speed,Thermolyne). The resulting conjugate was assigned identifier “pz041701.”

The conjugate mixture was purified using gel filtration chromatography.A size exclusion chromatography method was developed for analyzing thereaction mixtures, and the final products. SDS-PAGE analysis was alsoused for the characterization of the samples.

Conjugate Characterization. The resulting conjugate mixture, prior topurification, was a mixture of Factor VII PEG-monomer (or 1-mer), dimer(or 2-mer) and trimer (or 3-mer), corresponding to the identifiers“pz041701 (low),” “pz041701 (medium),” and “pz041701 (high)”respectively, as determined by SEC. That is to say: “pz041701 (low)”corresponds to mostly Factor VIII mono-PEGylated species or Factor VIIIhaving one PEG moiety attached thereto; “pz041701 (medium)” correspondsto primarily Factor VIII di-PEGylated species, that is to say, FactorVIII having two PEG moieties attached thereto; and “pz041701 (high)”corresponds mostly to Factor VIII having three PEG moieties attachedthereto. The corresponding SEC plots are shown in FIGS. 1 and 2. FIG. 1shows the SEC plot corresponding to fractions collected upon SEC of theFactor VIII reaction mixture. According to the size exclusionchromatography (SEC) results, the PEGylation yield of mPEG-SPA-30K-FVIII(pz041701 low) was ˜39%. The PEGylation yield of mPEG30-SPA-30K-FVIII(pz041701 medium) was ˜32%, and the PEGylation yield ofmPEG-SPA-30K-FVIII (pz041701 high) was ˜11%, with percentages based uponrelative amounts compared to all species present in the resultingreaction mixture. The conjugate mixture was further purified by FPLC andanalyzed by SDS-PAGE.

Example 7 Conjugation of B-Domain Deleted Factor VIII with mPEG-MAL, 20K

Prior to the conjugation, a buffer exchange for B-domain deleted FactorVIII (Factor VIII) was performed to replace histidine with HEPES.

mPEG-MAL, 20K, stored at −20° C. under argon, was warmed to ambienttemperature. The warmed mPEG-MAL reagent (4.4 mg) was dissolved in 0.044ml of HEPES buffer [50 mM HEPES, 0.15 M NaCl, 4.0 mM CaCl₂, 0.01% (w/v)TWEEN 80™ (Polyoxyethylene 20 sorbitan monooleate), pH 7.0] to make a10% mPEG-MAL solution. The mPEG-MAL solution was quickly added to 4 mlof Factor VIII solution [0.4324 mg/ml in 50 mM HEPES, 0.5 M NaCl, 4 mMCaCl₂, 0.1% (w/v) PEG 3,350, 0.01% (w/v) TWEEN 80™ (Polyoxyethylene 20sorbitan monooleate), pH 7.0] and mixed well. After 30 minutes ofreaction at room temperature, the reaction vial was transferred to thecold room (4° C.), and another 0.044 ml of mPEG-MAL solution was addedto the reaction mixture, followed by the addition of three more aliquotsof 0.044 ml of mPEG-MAL solution over the course of two hours. The pHwas determined (pH 7.0±0.2). The molar ratio of mPEG-MAL to protein was100:1. The final mPEG-MAL concentration was 5.213 mg/ml, and the finalFactor VIII concentration was 0.410 mg/ml. The reaction was allowed toproceed overnight at 4° C. on Rotomix (slow speed, Thermolyne). Theconjugate was assigned identifier “pz061201.”

The conjugate mixture was purified using gel filtration chromatography.A size exclusion chromatography method was developed for analyzing thereaction mixtures, and the final products. SDS-PAGE analysis was alsoused for the characterization of the samples.

CONJUGATE CHARACTERIZATION (Mono-PEGylated product). According to thesize exclusion chromatography (SEC) results, the PEGylation yield ofmonoPEGylated conjugate (1-mer) was ˜33% (FIG. 3). The Factor VIIIconjugate mixture fractions were combined and purified by FPLC, and thenfurther purified by gel filtration chromatography. The pz061201 finalproduct was analyzed by both SDS-PAGE and SEC, and the purity of the“pz061201” product was determined to be approximately 94% Factor VIIIPEG monomer (i.e., monopegylated Factor VIII), with ˜6% Factor VIII PEGhigh-mers (FIG. 4).

Example 8 Conjugation of B-Domain Deleted Factor VIII with mPEG-SMB, 30K

Prior to conjugation, a buffer exchange for B-domain deleted Factor VIII(Factor VIII) was performed to replace histidine with HEPES.

mPEG-SMB, 30K, stored at −20° C. under argon, was warmed to ambienttemperature. The warmed mPEG-SMB (6.5 mg) was dissolved in 0.065 ml of 2mM HCl to form a 10% mPEG-SMB solution. The mPEG-SMB solution wasquickly added to 4 ml of Factor VIII solution [0.435 mg/ml in 50 mMHEPES, 0.5 M NaCl, 5.0 mM CaCl₂, 0.1% (w/v) PEG 3,350, 0.01% (v/v) Tween80, pH 7.0] and mixed well. After 30 minutes of reaction at roomtemperature, the reaction vial was transferred to a cold room (4° C.).The pH was determined (pH 7.0±0.2). The molar ratio of mPEG-SMB toprotein was 20:1. The final mPEG-SMB concentration was 1.599 mg/ml, andthe final Factor VIII concentration was 0.428 mg/ml. The reaction wasallowed to proceed for approximately 48 hrs at 4° C. on Rotomix (slowspeed, Thermolyne), and was then quenched by the addition of acetic acid(99.7+%) to lower the pH to 6.0±0.3. The conjugate was assignedidentifier “pz082501.”

The conjugate mixture was purified using gel filtration chromatography.A size exclusion chromatography method was developed for analyzing thereaction mixtures, and the final products. SDS-PAGE analysis was alsoused for the characterization of the samples.

CONJUGATE CHARACTERIZATION: The mixture designated “pz082501” resultedfrom the PEGylation of Factor VIII with mPEG-SMB30K at pH 7.0±0.2. Theconjugate mixture was purified and analyzed by SEC. Based upon the sizeexclusion chromatography (SEC) results, the PEGylation yield ofpz082501, monoPEGylated conjugate (Factor VIII 1-mer) was ˜41% (FIG. 5).The product mixture was further purified by FPLC and analyzed bySDS-PAGE and SEC. Characterization of the purified Factor VIII PEGconjugate product, pz082501, was ˜95% mono-conjugated PEG Factor VIIIwith ˜5% higher-mers.

Example 9 Conjugation of B-Domain Deleted Factor VIII with mPEG-OPSS,10K

Prior to conjugation, a buffer exchange for B-domain deleted Factor VIII(Factor VIII) was performed to replace histidine with HEPES.

mPEG-OPSS, 10K, stored at −20° C. under argon, was warmed to ambienttemperature. mPEG-OPSS (1.2 mg) was dissolved in 0.012 ml of H₂O to makea 10% mPEG-OPSS solution. The mPEG-OPSS solution was quickly added to0.5 ml of Factor VIII solution [0.398 mg/ml in 50 mM Histidine, 0.5 MNaCl, 4.0 mM CaCl₂, 0.1% (w/v) PEG 3,350, 0.01% (w/v) TWEEN 80™(Polyoxyethylene 20 sorbitan monooleate), pH 6.7] and mixed well. After30 minutes of reaction at room temperature, the reaction vial wastransferred to a cold room (4° C.). The pH was determined (pH 6.7±0.2).The molar ratio of mPEG-OPSS-10K to protein was 100:1. The finalmPEG-OPSS concentration was 2.344 mg/ml, and the final Factor VIIIconcentration was 0.389 mg/ml. The reaction was allowed to proceedovernight at 4° C. on Rotomix (slow speed, Thermolyne).

The conjugate mixture was purified using gel filtration chromatography.A size exclusion chromatography method was developed for analyzing thereaction mixtures, and the final products, SDS-PAGE analysis was alsoused for the characterization of the samples. The pegylation results andyields using the mPEG-OPSS reagent were similar to those in Example 7,which employed the mPEG-MAL reagent having a molecular weight of 20K.

Example 10 Conjugation of B-Domain Deleted Factor VIII with mPEG-MAL,30K

Prior to conjugation, a buffer exchange for B-domain deleted Factor VIII(Factor VIII) was performed to replace histidine with HEPES.

mPEG-MAL, 30K, stored at −20K ° C. under argon, was warmed to ambienttemperature. The warmed mPEG-MAL (1.0 mg) was dissolved in 0.010 ml ofHEPES buffer [50 mM HEPES, 0.15 M NaCl, 4.0 mM CaCl₂, 0.01% (w/v) TWEEN80™ (Polyoxyethylene 20 sorbitan monooleate), pH 7.0] to make a 10%mPEG-MAL solution. The mPEG-MAL solution was quickly added to 0.5 ml ofFactor VIII solution [0.447 mg/ml in 50 mM HEPES, 0.5 M NaCl, 4 mMCaCl₂, 0.1% (w/v) PEG 3,350, 0.01% (w/v) TWEEN 80™ (Polyoxyethylene 20sorbitan monooleate), pH 7.0] and mixed well. After 30 minutes ofreaction at room temperature, the reaction vial was transferred to acold room (4° C.), and another 0.010 ml of mPEG-MAL solution was addedto the reaction mixture, followed by the addition of three more aliquotsof 0.010 ml of mPEG-MAL solution over the course of two hours. The pHwas determined (pH 7.0±0.2). The molar ratio of mPEG-MAL to protein was100:1. The final mPEG-MAL concentration was 9.091 mg/ml, and the finalFactor VIII concentration was 0.406 mg/ml. The reaction was allowed toproceed overnight at 4° C. on Rotomix (slow speed, Thermolyne).

The conjugate mixture was purified using gel filtration chromatography.A size exclusion chromatography method was developed for analyzing thereaction mixtures, and the final products. SDS-PAGE analysis was alsoused for the characterization of the samples. The PEGylation results andyields using the mPEG-MAL reagent having a molecular weight of 30 K weresimilar to those in Example 7, which employed the mPEG-MAL reagenthaving a molecular weight of 20K.

Example 11 Conjugation of B-Domain Deleted Factor VIII withmPEG-Butyr-ALD, 30K

Prior to conjugation, a buffer exchange for B-domain deleted Factor VIII(Factor VIII) was performed to replace histidine with HEPES.

mPEG-Butyr-ALD, 30K (shown above), stored at −20° C. under argon, waswarmed to ambient temperature. The warmed mPEG-Butyr-ALD (3.8 mg) wasdissolved in 0.038 ml of H₂O to make a 10% mPEG-Butyr-ALD solution. ThemPEG-Butry-ALD solution was quickly added to 0.5 ml of Factor VIIIsolution [0.400 mg/ml in 50 mM HEPES, 0.5 M NaCl, 5 mM CaCl₂, 0.1% (w/v)PEG 3,350, 0.01% (v/v) TWEEN 80™ (Polyoxyethylene 20 sorbitanmonooleate), pH 7.0] and mixed well. After 15 minutes, 0.060 ml of 10 mMsodium cyanoborohydride solution was added. The pH was determined (pH7.0±0.2). The molar ratio of mPEG-Butyr-ALD to protein was 100:1. Thefinal mPEG-Butyr-ALD concentration was 6.355 mg/ml. The final FactorVIII concentration was 0.334 mg/ml, and the final concentration ofNaCNBH₃ was 1.003 mM. The reaction was allowed to proceed for 5 hours atroom temperature, and then, overnight at 4° C. on Rotomix (slow speed,Thermolyne).

The conjugate mixture was purified using gel filtration chromatography.A size exclusion chromatography method was developed for analyzing thereaction mixtures, and the final products. SDS-PAGE analysis was alsoused for the characterization of the samples. The yield of Factor VIIImono-PEG conjugate was approximately 20%.

Example 12 In-Vitro Activity of Exemplary Factor VIII-PEG Conjugates

The in-vitro activities of the Factor VIII-PEG conjugates described inExamples 6, 7, and 8 were determined. All of the Factor VIII conjugatestested were bioactive.

1. A composition comprising a plurality of conjugates, each conjugatecomprising one, two or three water-soluble polymers each covalentlyattached to a Factor VIII moiety polypeptide via a hydrolytically stablelinkage, wherein: (i) the Factor VIII polypeptide is selected from thegroup consisting of Factor VIII, Factor VIIIa, Factor VIII:C, FactorVIII:vWF and B-domain deleted Factor VIII; (ii) the water-solublepolymer is selected from the group consisting of a poly(alkyleneglycol), a poly(oxyethylated polyol), a poly(olefinic alcohol), apoly(vinylpyrrolidone), a poly(hydroxyalkylmethacrylamide), apoly(hydroxyalkylmethacrylate), a poly(saccharide), a poly(α-hydroxyacid), a poly(vinyl alcohol), a polyphosphazene, a polyoxazoline, apoly(N-acryloylmorpholine), and combinations thereof; and (iii) thecomposition is bioactive, comprising an in-vitro activity of at least15% compared to that of a Factor VIII polypeptide composition inunconjugated form.
 2. The composition of claim 1, having an in-vitroactivity of at least 25% compared to the same Factor VIII polypeptidecomposition in unconjugated form.
 3. The composition of claim 1, havingan in-vitro activity of at least 40% compared to the same Factor VIIIpolypeptide composition in unconjugated form.
 4. The composition ofclaim 1, wherein each conjugate is comprised of the same Factor VIIIpolypeptide.
 5. The composition of claim 1, comprising a plurality ofconjugates wherein the conjugates each have one or two water-solublepolymers covalently attached to the Factor VIII polypeptide.
 6. Thecomposition of claim 1, wherein the conjugates each have onewater-soluble polymer covalently attached to the Factor VIIIpolypeptide.
 7. The composition of claim 1, wherein the conjugates eachhave two water-soluble polymers covalently attached to the Factor VIIIpolypeptide.
 8. The composition of claim 1, wherein the conjugates eachhave three water-soluble polymers covalently attached to the Factor VIIIpolypeptide.
 9. The composition of claim 4, wherein the water-solublepolymer is covalently attached to the Factor VIII polypeptide via alinkage selected from amide, secondary amine, carbamate, thioether, anddisulfide.
 10. The composition of claim 9, wherein the water-solublepolymer is covalently attached to the Factor VIII polypeptide in eachconjugate via an amide linkage.
 11. The composition of claim 1, whereineach water-soluble polymer in the composition has a nominal averagemolecular weight in a range of from greater than 5,000 Daltons to150,000 Daltons.
 12. The composition of claim 11, wherein each conjugatein the composition comprises the same water-soluble polymer.
 13. Thecomposition of claim 11, wherein the water-soluble polymer is apoly(alkylene glycol) or a polysaccharide selected from dextran andstarch.
 14. The composition of claim 13, wherein the poly(alkyleneglycol) is a poly(ethylene glycol).
 15. The composition of claim 14,wherein the poly(ethylene glycol) is terminally capped with anend-capping moiety selected from the group consisting of hydroxy,alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy,substituted alkynoxy, aryloxy and substituted aryloxy.
 16. Thecomposition of claim 14, wherein the poly(ethylene glycol) is terminallycapped with methoxy or hydroxy.
 17. The composition of claim 14, whereinthe poly(ethylene glycol) has a nominal average molecular weight in therange of from about 6,000 Daltons to about 100,000 Daltons.
 18. Thecomposition of claim 14, wherein the poly(ethylene glycol) has a nominalaverage molecular weight in the range of from about 10,000 Daltons toabout 85,000 Daltons.
 19. The composition of claim 18, wherein thepoly(ethylene glycol) has a nominal average molecular weight in therange of from about 20,000 Daltons to about 85,000 Daltons.
 20. Thecomposition of claim 14, wherein the poly(ethylene glycol) has a nominalaverage molecular weight selected from the group consisting of about6,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000Daltons, about 40,000 Daltons, about 50,000 Daltons, and about 60,000Daltons.
 21. The composition of claim 14, wherein each poly(ethyleneglycol) is linear.
 22. The composition of claim 14, wherein eachpoly(ethylene glycol) is branched.
 23. The composition of claim 14,wherein the plurality of conjugates each possess the same Factor VIIIpolypeptide, the same water-soluble polymer, and the same covalentattachment between the water-soluble polymer and the Factor VIIIpolypeptide.
 24. The composition of claim 14, wherein the Factor VIIIpolypeptide is selected from the group consisting of Factor VIII, FactorVIIIa, Factor VIII:C, and Factor VIII:vWF.
 25. The composition of claim14, wherein the Factor VIII polypeptide is B-domain deleted Factor VIII.26. The composition of claim 14, wherein the Factor VIII polypeptide isrecombinantly-derived.
 27. The composition of claim 14, wherein theFactor VIII polypeptide is blood-derived.